两种花吊丝竹叶片蛋白提取方法的2-DE比较  

Comparison of two protein extraction protocol of Dendrocalamus minor var. amoenus leaves with two-dimensional electrophoresis

作　　者：沈少炎 吴玉香[1,2] 荣俊冬[3,2] 何天友[1,2] 郑郁善[1,3,2] 

机构地区：[1]福建农林大学园林学院 [2]福建农林大学竹类研究所,福建福州350002 [3]福建农林大学林学院

出　　处：《福建农林大学学报（自然科学版）》2017年第1期50-57,共8页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建省科技重大专项资助(2013NZ0001)

摘　　要：以2年生花吊丝竹的扦插苗为研究材料,对2种方法(TCA/酚提结合法和PEG法)提取的蛋白质的2-DE差异进行研究.结果表明:PEG法的5个组分的条带差异明显,全蛋白得到了有效分离,在F3中富集到了2条高丰度蛋白带;2种方法的2-DE图谱的均一性分析发现,PEG法中F3富集了高丰度蛋白,提高了2-DE中蛋白质的检测率和分辨率,PEG法中F1-F5共检测的蛋白总数超过了1700个,但是相邻组存在一定的重叠率,而TCA丙酮/酚提结合法只检测到约571个蛋白点,5个组与NF平均有439个蛋白点匹配,达到77.9%以上;质谱鉴定了5种差异蛋白,生物信息学分析发现这5种蛋白是糖酵解、糖异生和ATP合成的重要辅酶.2种方法的比较发现,PEG法能够将花吊丝竹叶片全蛋白中高丰度蛋白单一富集在16%的组分中,从而显著提高了2-DE的分辨率,再通过质谱技术能够检测到更多的影响生物体生长发育的低丰度蛋白,所以PEG法是一种切实可行的且较为理想的蛋白质提取方法.Two years old cutting seedlings of Dendrocalamus minor var. amoenus w e r e u s e d to a n a l y z e protein spots a n d m a t c h i n g rates. The proteins extracted by TCA/phenol method and PEG methods ＂were separated using 2-DE technology. The result show,ed that the differences of protein bands between the 5 fractions generated by the PEG method were significant in the SDS-PAGE analysis and 2 highly abundant proteins bands enriched in F3,indicating an effectively separated total proteins. The homogeneity analysis of the 2-DE images by the 2 methods revealed that a highly abundant protein extracted by PEG method was enriched in fraction F3, which significantly increased the detection rate and resolution of 2-DE protein spots. In PEG method,the number of protein detected in F1-F5 was more than 1 700, but a certain degree of overlapping occurred between adjacent fractions. However,T C A / p h e n o l method could only detect about 571 protein spots,and on average 439 protein spots were matched between the 5 fractions and NF , resulting in a matching rate of more than 77.9%. Five different protein spots between PEG method and TCA acetone/phenol were i-dentified by MALDI-TOF MS. Referring to database matching and bioinformatic analysis,these 5 proteins turned out to be essential coenzyme for plant involved in glycolysis,gluconeogenesis and ATP synthesis. To summarize,traditional TCA acetone/phenol meth-od was unable to detect low-abundant proteins in Dendrocalamus minor var. amoenus leaves. H o w e v e r, the total proteins c o u l d b e separated into 5 fractions by PEG method and 16% of proteins （ F3 ） could be enriched in a single fraction of high-abundant pro- teins,which resulted in higher detection rate and significantly improved resolution of 2-DE. More low-abundant proteins that affect growth and development of organism can be detected by mass spectrometry. The PEG method is a practical and ideal proteomics re-search method for bamboos or other plants.

关 键 词：花吊丝竹 TCA/酚提结合法 PEG法 双向电泳 生物信息学 

分 类 号：S718.43[农业科学—林学]